CN109637590A - A kind of microsatellite instability detection system and method based on gene order-checking - Google Patents
A kind of microsatellite instability detection system and method based on gene order-checking Download PDFInfo
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Abstract
A kind of microsatellite instability detection system and method based on gene order-checking, microsatellite detection site selection: according to the sequencing data to certain tumor sample, effective detection site is selected, the corresponding single instable threshold value of microsatellite locus of effective detection site and the evaluation criterion of certain tumor sample microsatellite instability are calculated；According to the corresponding single instable threshold value of microsatellite locus of effective detection site and the evaluation criterion of certain tumor sample microsatellite instability, microsatellite instability detection is carried out to detection sample.The present invention does not depend on check sample, it is possible to reduce sampling is to give detected person's bring pain；A possibility that whole hereditary information in check sample containing testee, the present invention can be reduced without using check sample to testee's privacy leakage；The cost of detection can be reduced by not detecting check sample.Operation of the present invention is convenient, at low cost, with a high credibility.
Description
Technical field
The present invention relates to a kind of microsatellite instability detection methods, and in particular to a kind of micro- based on gene order-checking defends
Star unstability detection system and method.
Background technique
In recent years, human genome sequencing technologies are in disease, health, aging etc. using more and more extensive, with
The maturation of sequencing technologies, (Next Generation Sequencing, NGS, i.e. two generations are surveyed especially next-generation sequencing technologies
Sequence) gradually it is applied to clinical diagnosis.The invention of next-generation sequencing technologies is so that sequencing technologies realize automation high throughput two
A feature, so that sequencing price is lower and lower, up to the present about 1000 dollars of full-length genome numbers for being obtained with a people
According to.In addition to next-generation sequencing technologies, at present using PacBio and OxfordNanopore as the real-time single-molecule sequencing of representative, and
Unicellular sequencing also observes human genomic sequence information for the mankind on a microscopic level and provides more conveniences.
Microsatellite (Microsatellite is also Short tandem repeatSTR, Short Tandem Repeat, STR) refers generally to
Using 1-5 base as the repetition of basic unit in genome.When in cell with DNA mismatch repair system (MismatchRepair
System, MMR) relevant gene mutate or abnormal apparent modification and when inactivating, microsatellite instability will be generated
The phenotype of (Microsatellite Instability, MSI).A large number of studies show that the hair of microsatellite instability and tumour
Life is related, and plays an important role during the treatment of tumour and prognosis.
As China human mortality Aging Problem aggravates, the disease incidence of cancer is higher and higher.Immunization therapy is as a kind of individual character
The advantage that there are the tumor therapeuticing method of change the traditional therapies such as radiotherapy, chemotherapy not have is for example black in kinds of tumors at present
Melanoma illustrates powerful anti-tumor activity in the treatment of the solid tumors such as non-small cell lung cancer, kidney and prostate cancer, more
Kind immunotherapy of tumors drug, which has obtained U.S. Food and Drug Administration, to be ratified and is clinically applied.But
It is that immunotherapy medicaments are not all sensitive to all cancers, largely studies have shown that the cancer of the microsatellite instability positive
Patient has preferable outcome and longer life span after receiving immunization therapy.Meanwhile a large number of studies show that in Colon and rectum
Cancer, gastric cancer, in carcinoma of endometrium, the patient of the microsatellite instability positive is not suitable for using some chemotherapeutics, such as 5- fluorine urea
Pyrimidine, and more suitable for immunization therapy.Therefore the morning of cancer is sieved in the detection of this phenotype of microsatellite instability, treats and pre-
All have very important significance afterwards.
Microsatellite instability detection method depends on the prepared detection mark of U.S.'s tumor research in clinic at present
Standard, that is, detect two mononucleotides repeat site (BAT-25, BAT-26) and three dinucleotides repetition sites (D2S123,
D5S346, D17S250) totally five genome microsatellite locus stability.Then this method passes through electrophoresis by PCR amplification
Experimental comparison's tumor sample and normal control sample determine the steady of microsatellite locus in the copy number situation of target repeat region
It is qualitative.It can be by sample microsatellite instability shape according to the ratio of the total check bit points of unstability site Zhan in detection sample
State is determined as high unstable (MSI-H), low unstable (MSI-L) and stablizes (MSS) three kinds of states.This detection method check bit
Point is very few, and experimental program is complicated, and it is longer to expend the time.In the recent period to the gene order-checking data of cancer sample studies have shown that tool
There is the sample of MSI-L and MSS phenotype to be not significantly different, the method for PCR electrophoresis experiment can not obtain this conclusion.Although one
A little patents or document propose, increase MSI detection site and contrived experiment to increase the accuracy rate of detection, but this are based on
The detection method of PCR and electrophoresis experiment not can be carried out high throughput automated progress, while detection site increase will will increase experiment
Difficulty and cost, cannot tackle the problem at its root.The reality of this based on PCR and the detection method of electrophoresis experiment at the same time
Step complexity is tested, it is expensive, and normal tissue and tumor tissues is needed to compare.
Microsatellite instability is judged except through the detection to microsatellite locus, and there are also immune for the method for mainstream at present
The method of groupization, i.e., by the expression of gene relevant to mismatch repair system in antigen-antibody hybridization check tumour come really
Determine the state of microsatellite instability.Although this method sensitivity is higher, as above-mentioned experimental method, same test is multiple
Miscellaneous, repeatability is low, and normal sample is needed to compare.
Current some researchers judge MSI state using two generation sequencing datas, and work well, but experiment is set
Many defects are still had in meter and method, are specifically described as follows:
MSIseq(Ni Huang,M.,et al.MSIseq:Software for Assessing Microsatellite
Instability from Catalogs of Somatic Mutations.Scientific Reports 2015；5(1).)
It is that the variation for carrying out single nucleotide polymorphism (SNP) and small insertion and deletion (Indel) to sample using sequencing data detects, so
MSI state is judged using machine learning method according to variation testing result afterwards.Although this method does not need normal control sample
This, but need to carry out variation detection to sample, this will spend a large amount of computing resource and time, and this method is accurate
Property accuracy dependent on variation testing result, and the stability of genome mutation testing result is poor at present, especially micro-
The repeat region of this type of satellite.
MSIsensor(Niu,B.,et al.MSIsensor:microsatellite instability detection
using paired tumor-normal sequence data.Bioinformatics 2014；30(7):1015-1016.)
With MOSAIC (Hause, R.J., et al.Classification and characterization of
microsatellite instability across 18cancer types.Nat Med 2016；22(11):1342-
1350.) feature for directly extracting microsatellite on genome carries out the judgement of subsequent microsatellite stability, there is no computing resource and
The problem of time, accuracy rate is also relatively high, but normal sample is needed to compare, and this not only adds cost and can not obtain
It cannot be detected when obtaining normal sample, it is upper with significant limitation in application.
Summary of the invention
The purpose of the present invention is to provide a kind of microsatellite instability detection system and method based on gene order-checking.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of microsatellite instability detection system based on gene order-checking, including microsatellite detection site selecting module
With microsatellite instability detection module；
Wherein, the microsatellite detection site selecting module is used for according to the sequencing data to certain tumor sample, selection
Effective detection site calculates the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumour sample
The evaluation criterion of this microsatellite instability；Microsatellite instability detection module is used to select mould according to microsatellite detection site
The corresponding single instable threshold value of microsatellite locus of effective detection site that block obtains and certain tumor sample microsatellite
Instable evaluation criterion carries out microsatellite instability detection to detection sample.
A further improvement of the present invention lies in that the microsatellite detection site selecting module includes single microsatellite locus base
Because type extraction module, removal verification and measurement ratio be unsatisfactory for desired site module, the higher site module of removal crowd's diversity and
Remove the module in the small site of discrimination；
Wherein, the single microsatellite locus genotype extraction module is compared for pre-processing to sequencing data
To data, then comparison data is scanned, obtains the genotype of each microsatellite locus, the gene of each microsatellite locus
Type specifically includes the position that microsatellite locus place refers to genome, repetitive unit, and the number of repetition on reference genome is surveyed
Ordinal number detects the position repetitive unit number of repetition, corresponding read number and other optional informations in；
Removal verification and measurement ratio is unsatisfactory for desired site module and is used to carry out microsatellite locus gene to all training samples
Type extracts, and obtains the genotype of all training samples；Then filtering is unsatisfactory for the microsatellite locus of verification and measurement ratio requirement；
The higher site module of removal crowd's diversity is used for some microsatellite locus for some sample, uses microsatellite
Repetitive unit be averaged number of repetition instead of the microsatellite locus in the sample genotype；Then for each microsatellite locus,
Calculate the mean μ and standard deviation sigma of sample microsatellite genotype；The statistic being made of mean value and standard deviation is to microsatellite locus
Diversity is assessed；Assessment result is carried out according to diversity, and screens microsatellite locus using diversity；
The small site module of discrimination is removed for calculating the microsatellite in the microsatellite instability positive and negative sample
Site stability difference, then screening obtains effective detection site.
A kind of microsatellite instability detection method based on gene order-checking, including microsatellite instability detection method
It is broadly divided into the selection of microsatellite detection site and microsatellite instability detects two processes, specific as follows:
Step 1: microsatellite detection site selects: according to the sequencing data to certain tumor sample, selecting effective check bit
Point calculates the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumor sample microsatellite not
The evaluation criterion of stability；
Step 2: microsatellite instability detects: the effective detection site obtained according to step 1 is corresponding single micro- to be defended
The evaluation criterion of the instable threshold value of championship point and certain tumor sample microsatellite instability carries out detection sample micro-
The detection of satellite unstability.
A further improvement of the present invention lies in that selecting effective detection site, detailed process is as follows:
1) single microsatellite locus genotype is extracted: being pre-processed first to sequencing data, is obtained comparison data, then
Comparison data is scanned, the genotype of each microsatellite locus is obtained, the genotype of each microsatellite locus specifically includes
Refer to the position of genome where microsatellite locus, repetitive unit, the number of repetition on reference genome is examined in sequencing data
Measure the position repetitive unit number of repetition, corresponding read number and other optional informations；
2) removal verification and measurement ratio is unsatisfactory for desired site: carrying out microsatellite locus genotype to all training samples and mentions
It takes, obtains the genotype of all training samples；Then filtering is unsatisfactory for the microsatellite locus of verification and measurement ratio requirement；
3) for some microsatellite locus of some sample, the sample is replaced with the microsatellite repetitive unit number of repetition that is averaged
In the microsatellite locus genotype；Then for each microsatellite locus, the mean μ and mark of sample microsatellite genotype are calculated
Quasi- difference σ；The statistic being made of mean value with standard deviation assesses the diversity of microsatellite locus；It is commented according to diversity
Estimate as a result, and screening microsatellite locus using diversity；
4) the microsatellite locus stability difference in the microsatellite instability positive and negative sample is calculated, is then screened
To effective detection site.
5. a kind of microsatellite instability detection method based on gene order-checking according to claim 4, special
Sign is, in step 2), filters microsatellite locus of the verification and measurement ratio less than 50%；
In step 3), the microsatellite locus of σ/μ > 1 is removed when screening microsatellite locus using diversity.
A further improvement of the present invention lies in that being calculated in the microsatellite instability positive and negative sample in step 4)
Microsatellite locus stability difference, then screening obtains effective detection site detailed process is as follows:
4.1) evaluation method of single microsatellite stability is determined, detailed process is as follows:
When defect occurs for intracellular mismatch repair system, mistake occurs in duplication and accumulates by intracellular DNA, such as
Fruit template strand occurs to fold the variation that the same position DNA is then deleted in then subsequent progeny cell, if synthesis chain
It folds, then insertion variation can occur for the same position DNA in the progeny cell of back；In order to detect microsatellite instability
Property, it is a multinomial distribution by the process simulation of DNA cloning；Detailed process is as follows:
Assuming that for DNA during synthesis, each base or repetitive unit synthesis process are independent in microsatellite region
, Fold will not occur simultaneously for the same position, template strand and synthesis chain；Mould when each base or each unit replicate
Plate chain folding is event D, and the probability occurred is p；Synthesis chain folding is event I, probability q；The situation template chain and conjunction
Chaining is not folded into event N, probability 1-p-q；Assuming that DNA microsatellite region base or again in a replication process
Multiple unit number is L, and duplication produces m copy altogether, and the number that wherein event D occurs is d, and the number that event I occurs is i,
The probability that event N occurs is n, then d+i+n=mL；
Had based on above-mentioned hypothesis:
P in above formula, q pass through d, i, n estimation；
For each detected microsatellite locus, the stability of single microsatellite is measured according to p and/or q；
4.2) according to the single microsatellite locus method for estimating stability of step 4.1), microsatellite position in all samples is calculated
The stability of point；For each microsatellite locus, examine the site stability whether in microsatellite using non-parametric test method
There is significant difference in negative sample and in positive sample；Then examine all microsatellite locus, removal in negative sample and
The microsatellite locus being not significantly different in positive sample obtains effective detection site.
A further improvement of the present invention lies in that calculating the corresponding single microsatellite locus of effective detection site in step 1
Detailed process is as follows for instable threshold value: each effective detection site is directed to, using order is micro- really in step 4.1)
The evaluation method of satellite stability calculates the stability in microsatellite instability negative sample, and the mean value of computational stability with
Variance, using the sum of stability mean value and three times variance as the threshold value of the microsatellite locus stability.
A further improvement of the present invention lies in that determining the evaluation of certain tumor sample microsatellite instability in step 1
Detailed process is as follows for standard: according to the threshold value of the corresponding microsatellite locus stability of effective detection site, to each in sample
The stability of microsatellite is judged, then according to the stability of effective detection sites all in sample to sample overall stability
It is assessed, determines that sample entirety microsatellite instability detects evaluation criterion.
A further improvement of the present invention lies in that calculating the stabilization of all effective detection sites in each sample in step 1
Property, and calculate unstable anchor point and account for all ratios for detecting effective site, then according to ratio to sample microsatellite instability
Property judged, being chosen at value when sensibility and specificity reaches maximum is commenting for certain tumor sample microsatellite instability
Price card is quasi-, positive for microsatellite instability when test sample is greater than the evaluation criterion, is otherwise microsatellite instability yin
Property.
A further improvement of the present invention lies in that detailed process is as follows for step 2: being had first according to microsatellite instability
The set of detection site is imitated, sequencing data is scanned, the genotype distribution of each microsatellite locus is obtained, then calculates individually micro- defend
The stability of championship point and according to the stability of the single microsatellite of the corresponding threshold decision of the stability of single microsatellite locus, most
The evaluation criterion of certain tumor sample microsatellite instability afterwards judges sample microsatellite instability, obtains sample
Whole microsatellite stability result.
Compared with prior art, the invention has the benefit that existing microsatellite detection method requires greatly control sample
This, the present invention does not need check sample, it is possible to reduce the limitation of microsatellite detection, and have the advantage that first, it compares
Sample sampling generally requires to obtain testee's blood or Carcinoma side normal tissue, the present invention do not depend on check sample, can subtract
Detected person's bring pain is given in few sampling；Second, whole hereditary information containing testee in check sample, the present invention is not
A possibility that being reduced using check sample to testee's privacy leakage；Third, inspection can be reduced by not detecting check sample
The cost of survey.Since the present invention can obtain the heredity letter of testee's cancerous tissue using two mature at present generation sequencing technologies
Breath, it is easy to operate, it is at low cost, it is with a high credibility.
Further, single microsatellite locus method for estimating stability of the invention is from the principle of microsatellite mechanism
Modeling, the genesis mechanism of microsatellite instability can be explained from principle, can more accurately estimate single microsatellite
The stability in site.
Further, the present invention has very high accuracy in microsatellite instability context of detection, and currently based on PCR
There is very high consistency with the result of the clinical goldstandard method of electrophoresis experiment.
Further, the present invention is assessed by single microsatellite stability, can be from the principle of microsatellite instability
The stability of microsatellite locus is described.Compared with current single microsatellite instability detection method, the model in the present invention is not
Control group data are needed, and are had very strong explanatory.
Further, the present invention is selected by microsatellite instability detection site selection method using Principle of Statistics
Site relevant to microsatellite instability carries out microsatellite instability detection, is not having the case where control group, it is possible to reduce
Due to crowd's microsatellite diversity bring error, and reduce testing cost.
Further, the present invention utilizes the sequencing data of sample by microsatellite instability evaluation criterion method for building up
And corresponding microsatellite instability state learns microsatellite instability feature, and it is unstable to establish single microsatellite locus
The evaluation criterion of qualitative evaluation standard and sample microsatellite instability.
Detailed description of the invention
Fig. 1 is overall work exemplary diagram of the present invention.
Fig. 2 is single microsatellite locus Detection of Stability model.
Fig. 3 is the selection of microsatellite detection site, site and Almost Sure Sample Stability standard Establishing process figure.
Fig. 4 is that microsatellite genotype distributed data format stores example.
Fig. 5 is the non-dependent microsatellite instability detection schematic diagram of check sample.
Fig. 6 is DNA sample sequencing and data preprocessing operation process.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawing.
The present invention provides a kind of microsatellite instability detection system based on gene order-checking, including microsatellite check bit
Point selection module and microsatellite instability detection module；
Wherein, the microsatellite detection site selecting module is used for according to the sequencing data to certain tumor sample, selection
Effective detection site calculates the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumour sample
The evaluation criterion of this microsatellite instability；Microsatellite instability detection module is used to select mould according to microsatellite detection site
The corresponding single instable threshold value of microsatellite locus of effective detection site that block obtains and certain tumor sample microsatellite
Instable evaluation criterion carries out microsatellite instability detection to detection sample.
Specifically, the microsatellite detection site selecting module includes single microsatellite locus genotype extraction module, goes
Except verification and measurement ratio is unsatisfactory for desired site module, the higher site module of removal crowd's diversity and removes the small position of discrimination
The module of point；
Wherein, the single microsatellite locus genotype extraction module is compared for pre-processing to sequencing data
To data, then comparison data is scanned, obtains the genotype of each microsatellite locus, the gene of each microsatellite locus
Type specifically includes the position that microsatellite locus place refers to genome, repetitive unit, and the number of repetition on reference genome is surveyed
Ordinal number detects the position repetitive unit number of repetition, corresponding read number and other optional informations in；
Removal verification and measurement ratio is unsatisfactory for desired site module and is used to carry out microsatellite locus gene to all training samples
Type extracts, and obtains the genotype of all training samples；Then filtering is unsatisfactory for the microsatellite locus of verification and measurement ratio requirement；
The higher site module of removal crowd's diversity is used for some microsatellite locus for some sample, uses microsatellite
Repetitive unit be averaged number of repetition instead of the microsatellite locus in the sample genotype；Then for each microsatellite locus,
Calculate the mean μ and standard deviation sigma of sample microsatellite genotype；The statistic being made of mean value and standard deviation is to microsatellite locus
Diversity is assessed；Assessment result is carried out according to diversity, and screens microsatellite locus using diversity；
The small site module of discrimination is removed for calculating the microsatellite in the microsatellite instability positive and negative sample
Site stability difference, then screening obtains effective detection site.
The present invention provides a kind of microsatellite instability detection method based on the sequencing of two generations, and it is non-dependent that packet expands check sample
Microsatellite instability detection, establish single microsatellite stability assessment models, the selection of microsatellite instability detection site,
And microsatellite instability evaluation criterion is established.
As shown in Figure 1, microsatellite instability detection method of the invention be broadly divided into microsatellite detection site selection and
Microsatellite instability detects two processes,
Step 1: microsatellite detection site selects: according to the sequencing data to certain tumor sample, selecting effective check bit
Point calculates the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumor sample microsatellite not
The evaluation criterion of stability；For same tumour, the step for only need to carry out it is primary.
As shown in figure 3, the selection of microsatellite instability detection site by with the sample of known microsatellite instability state and
Its tumour sequencing data is input, obtains microsatellite locus relevant to microsatellite instability state.Microsatellite instability
State can be examined quasi- by the prepared examination criteria of U.S.'s tumor research (PCR and electrophoresis method) or MSIsensor etc.
The true higher method of rate obtains.Sequencing data can be full-length genome, the sequencing of full exon group or target area.According to
To the sequencing data of certain tumor sample, selecting effective detection site, detailed process is as follows:
1) single microsatellite locus genotype is extracted: being pre-processed first to sequencing data, is obtained the comparison of high quality
Data, the storage format of comparison data can be bam format or other comparison data storage formats, then to comparison data into
Row scanning, obtains the genotype of each microsatellite locus, the genotype of each microsatellite locus specifically includes microsatellite locus institute
In the position of reference genome, repetitive unit, the number of repetition on reference genome detects position weight in sequencing data
Multiple unit number of repetition, corresponding read number and other optional informations.Fig. 4 provides a kind of microsatellite locus genotype storage format
Example.
2) removal verification and measurement ratio is unsatisfactory for desired site: carrying out microsatellite locus genotype to all training samples and mentions
It takes, obtains the genotype of all training samples.The verification and measurement ratio of each microsatellite locus is by detecting the site in training sample
The ratio of the number of sample number and all training samples determines.The verification and measurement ratio of microsatellite locus is to measure the microsatellite region working as
The ability of sequencing data is obtained under preceding sequencing approach (full-length genome, full exon group, one of target area sequencing), if
Microsatellite locus verification and measurement ratio is too low, shows the sequencing data that this microsatellite locus can not be obtained in numerous samples.In order to
Guarantee that the microsatellite locus number of final choice is enough, and guarantees that selected microsatellite locus has high inspection in all samples
Survey rate, user can weigh the two pros and cons, choose the microsatellite locus that suitable threshold filtering is unsatisfactory for verification and measurement ratio requirement.Such as it goes
Site except verification and measurement ratio less than 50%.
3) the higher site of crowd's diversity is removed: studies have shown that some microsatellites have very high multiplicity in crowd
Property, it can be used as the mark of identification, be widely used in medical jurisprudence.In the present invention, it needs to select in genome
Conservative microsatellite locus relatively, is analyzed.
For some microsatellite locus of some sample, replaced in the sample with the microsatellite repetitive unit number of repetition that is averaged
The genotype of the microsatellite locus.For each microsatellite locus, the mean μ and standard deviation sigma of sample microsatellite genotype are calculated.
The statistic being made of mean value with standard deviation assesses the diversity of microsatellite locus.According to real data feature, most
Retain in the enough situations of number of loci eventually, user weighs the two and determines a threshold value, the high microsatellite locus of removal diversity.
The diversity of microsatellite is such as evaluated with σ/μ, σ/μ shows that more greatly the diversity of microsatellite is higher, can remove σ/μ > 1
Microsatellite locus.
4) the small site of removal discrimination: discrimination refers to the contribution that microsatellite locus detects microsatellite instability.This
Removal small site of microsatellite locus stability difference in the microsatellite instability positive and negative sample, is had in invention
Imitate detection site；Specifically includes the following steps:
4.1) evaluation method of single microsatellite stability is determined, detailed process is as follows:
As shown in Fig. 2, when defect occurs for intracellular mismatch repair system, (defect here is referred to and mispairing reparation
System mutates or inactivates to relevant gene), mistake occurs in duplication and occurs and accumulates by intracellular DNA, especially
, template chain folding or synthesis chain folding can occur in microsatellite region shown in Fig. 2.If the raw folding of template running fire
The variation that DNA is then deleted the same position in subsequent progeny cell, if synthesis chain folds, back filial generation
Insertion variation can occur for the same position DNA in cell.When DNA is expanded in vitro, can equally occur template chain folding or
Person synthesizes chain folding.In order to detect the function i.e. microsatellite instability of mismatch repair system, it is by the process simulation of DNA cloning
One multinomial distribution.Detailed process is as follows:
Assuming that for DNA during synthesis, each base or repetitive unit synthesis process are independent in microsatellite region
, the Fold in Fig. 2 will not occur simultaneously for the same position, template strand and synthesis chain.Each base or each unit
Template strand is folded into event D when duplication, and the probability occurred is p, i.e., the probability that the position is deleted is p；Synthesize chain folding
For event I, probability q, i.e., the probability that the position is inserted into is q.The situation template chain and synthesis chain do not fold
That is it does not morph as event N, probability 1-p-q the position.Assuming that DNA in a replication process microsatellite region base or
Repetitive unit number is L, and duplication produces m copy altogether, and the number that wherein event D occurs is d, and the number that event I occurs is
The probability that i, event N occur is n, then d+i+n=mL.
Had based on above-mentioned hypothesis:
P in above formula, q can be estimated by d, i, n.Estimation method, can use Estimation of Mean, Bayesian Estimation etc. into
Row, details are not described herein again, can be any optional multinomial distribution method for parameter estimation.
For each detected microsatellite locus, its stability can be measured according to the probability of p and/or q.
4.2) according to the single microsatellite locus method for estimating stability of step 4.1), microsatellite position in all samples is calculated
The stability of point.For each microsatellite locus, examine the site stability whether in microsatellite using non-parametric test method
(statistical check p-value value has less than 0.05,0.01 or 0.1 to be shown in negative sample and with significant difference in positive sample
Write difference).All microsatellite locus are examined, the microsatellite position being not significantly different in negative sample and positive sample is removed
Point obtains effective detection site.
Such as the stability of single microsatellite can be measured with the probability that microsatellite is deleted, it is examined with rank sum test
Survey whether microsatellite locus stability has significant difference in microsatellite instability positive sample and negative sample.
The microsatellite locus obtained after step 4) can be used as the detection site of microsatellite instability or for most
Whole effective detection site.
5) the single instable threshold value of microsatellite locus is calculated:
Sample single locus stable calculation: for each detection site as microsatellite instability, micro- defend is calculated
Stability in star unstability negative sample, and its mean value and variance are calculated, the microsatellite position is determined using mean value and variance
The threshold value of point stability.
For example, the stability for the Probability Detection microsatellite locus deleted with microsatellite locus, by stability mean value with
Threshold value of the sum of three times variance as stability, i.e., determine the stabilization of single microsatellite locus in test sample according to the threshold value
Property.
6) it determines the evaluation criterion of certain tumor sample microsatellite instability: being selected according to above-mentioned steps 4 all
Detection site and its corresponding stability threshold, the stability of microsatellite each in sample is judged, then basis
The stability of all effective detection sites assesses sample overall stability in sample, determines sample entirety microsatellite instability
Qualitative detection evaluation criterion.It is described as follows:
The stability of all detection sites in each sample is calculated, and calculates unstable anchor point and accounts for and all detect significance bit
The ratio of point.Then sample microsatellite instability is judged according to ratio, the bigger expression sample microsatellite of ratio is more not
Stablize, the value being chosen at when sensibility and specificity reaches maximum is its threshold value standard.When test sample is greater than the threshold value
It is otherwise microsatellite instability feminine gender for the microsatellite instability positive.
Step 2: microsatellite instability detects: being surveyed to the microsatellite locus of 4 final choice of process in step 1)
Sequence, and microsatellite instability detection is carried out to detection sample.Detailed process is as follows:
In microsatellite locus selection course in step 1), step 4 obtains the effective check bit of microsatellite instability
Point, step 5 obtain the stability threshold in site in step 4, and sample microsatellite instability threshold value is obtained in step 6.It is right
In a certain particular cancers type, specific sequencing strategy (full-length genome, in the sequencing of full exon group or target area
It is a kind of), step 1) only needs to carry out primary.
For new detection sample, microsatellite instability detection process is as follows:
As shown in figure 5, sequencing data is scanned according to the effective detection site set of microsatellite instability in step 4 first,
The genotype distribution for obtaining each microsatellite locus, calculates the stability of single microsatellite locus and according to corresponding stability threshold
Value judges the stability of single microsatellite.After getting single microsatellite locus stability information, according to process 6 in step 1)
Middle sample microsatellite instability calculation method and sample microsatellite instability threshold value carry out sample microsatellite instability
Judgement, generates final microsatellite instability examining report.Information of the report comprising detection sample, the microsatellite locus letter of detection
Breath, effective microsatellite locus, the stability result of each microsatellite locus, the microsatellite stability result etc. of sample entirety.
It is below specific embodiments of the present invention.
1) DNA sequencing sample extraction and sequencing
Referring to Fig. 6, including tumor sample DNA extraction and purification, sequencing library building, the control of sequencing library quality, upper machine
Sequencing.Detailed description are as follows:
Tumor sample DNA extraction and purification: user can puncture the tumor sample of acquisition to the tumor tissues of operation excision
Or the tumour cell containing inhereditary material that other modes obtain carries out the detection and analysis of satellite unstability, it can also be to tumour
Cell line extracts DNA and carries out microsatellite instability detection.
Sequencing library building: according to user demand, building DNA sequencing library can be carried out to the DNA after extraction purification.Root
According to other needs of user, the library of full-length genome, full exon group or specific target areas sequencing can be constructed, this patent relates to
And method there is no particular/special requirement to the region of sequencing.
The control of sequencing library quality: quality control is carried out to the library that previous step is established, judges that can DNA sequencing library into
On row machine be sequenced, if cannot if need to rebuild gene library.
Upper machine sequencing: DNA is sequenced in this step, and Illumina, BGI can be used, and Pacbio, Nanopare etc. are surveyed
Sequence instrument is sequenced and is Base Calling.
2) sequencing data pre-processes
It is controlled including Raw data quality, with reference genome alignment, removal repeats read, the control of comparison data quality etc.
Process is described as follows:
Raw data quality control is the information examined sequencing data quality whether to meet the requirements and count sequencing sample, and
Do certain processing.The step in need to remove in sequencing data may joint sequence in mixed sequencing library, and go
Except sequencing quality is lower than the base or read of Q30.After to data filtering, the G/C content distribution to sample is needed, is read
Segment length distribution, Insert Fragment length etc. is analyzed, high-quality using these if filtered sequencing data requisite quality
The data of amount carry out next step analysis.
Refer to that the sequencing data by the high quality that previous step obtains compares on reference genome with reference genome alignment,
To obtain location information of each read on reference genome.Comparing software can select according to microarray dataset difference, such as
Two generations, which were sequenced, may be selected BWA, and Bowtie etc., Pacbio and Nanopore can choose nimimap2, ngmlr etc..
Removal repetitive sequence, which refers to, obtains each read behind the position on reference genome, and filtering is because of the sequencing of two generations
Polymerase chain reaction leads to same significantly the expanding for segment and the error that introduces in the process, such as can be used picard or
The softwares such as bammarkduplicates.
The control of sequencing data quality, which refers to, carries out quality control to the file after previous step filtration treatment, calculates it in reference base
Because of the coverage in group, the microsatellite locus number of covering, to determine whether to carry out subsequent analysis.
3) microsatellite detection site selects
The present invention selects in U.S.'s Oncogenome route map (The Cancer Genome Atlas, TCGA) database
Cancer sample carry out site selection.By taking colorectal cancer as an example, we have obtained microsatellite instability in TCGA database
Property result the full exon of tumor sample of 588 (MSI-L/MSS has 510, and MSI-H has 78) colorectal cancers number is sequenced
According to wherein 137 (MSI-L/MSS has 109, and MSI-H has 28) progress microsatellite detection sites select and subsequent mark for selection
Standard is established, and referred to as training sample set, remaining sample are known as test sample collection.
Genotype extraction is carried out to 137 samples, and stability assessment is carried out to all sites of sample, is defended here with micro-
The probability that championship point is deleted represents the stability of single microsatellite.It is detected altogether about in all samples after above-mentioned steps
100000 or more microsatellite locus, remaining 12752 sites in the lower site of removal verification and measurement ratio, removal diversity are higher
After microsatellite locus, higher 6389, the site of conservative is obtained.It is selected by rank sum test in MSI-H sample and MSS/MSI-
Stability has the site of significant difference in L sample, and taking p value is 0.05, and finally totally 1567 sites are examined for microsatellite
It surveys.
4) single microsatellite instability threshold value and sample microsatellite instability evaluation criterion are established
It carries out single microsatellite stability standard according to MSI-L/MSS sample in the test sample in step 3) to establish, meter
The mean value and standard deviation of single microsatellite locus unstability evaluation index in MSI-L/MSS sample are calculated, with mean value and three times mark
Quasi- difference is the threshold value of single microsatellite instability.It is to think to test when the estimation of stability index of test sample is greater than the threshold value
The microsatellite locus is unstable in sample.
The stability of microsatellite detection site in all samples is calculated, and calculates unstable microsatellite locus and accounts for all detections
Microsatellite locus ratio, the stability of sample entirety microsatellite is assessed with this.Then according to ratio to sample microsatellite
Unstability is judged that the value being chosen at when sensibility and specificity reaches maximum is its standard.It is somebody's turn to do when test sample is greater than
As microsatellite instability is positive when threshold value.
5) microsatellite instability detection test
Choosing test sample concentrates 451 tumor samples to be tested
The standard for the colorectal cancer test sample microsatellite instability detection that 451 test samples are concentrated in TCGA database
True rate.Using the result of PCR goldstandard as legitimate reading, calculate obtain microsatellite instability detection method of the present invention and
The evaluation index of MSIsensor is as shown in table 1 below:
The evaluation index of table 1 microsatellite instability detection method and MSIsensor of the present invention
The present invention and PCR goldstandard method acquired results have 99.32% consistency, compared with MSIsensor, the present invention
Accuracy specificity, accuracy is all higher.
Claims (10)
1. a kind of microsatellite instability detection system based on gene order-checking, which is characterized in that including microsatellite check bit
Point selection module and microsatellite instability detection module；
Wherein, the microsatellite detection site selecting module is used for according to the sequencing data to certain tumor sample, and selection is effective
Detection site, calculates the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumor sample is micro-
The instable evaluation criterion of satellite；Microsatellite instability detection module according to microsatellite detection site selecting module for obtaining
The corresponding single instable threshold value of microsatellite locus of the effective detection site arrived and certain tumor sample microsatellite instability
Qualitative evaluation criterion carries out microsatellite instability detection to detection sample.
2. a kind of microsatellite instability detection system based on gene order-checking according to claim 1, feature exist
It include that single microsatellite locus genotype extraction module, removal verification and measurement ratio are discontented in, the microsatellite detection site selecting module
The module in the small site of site module, the higher site module of removal crowd's diversity and the removal discrimination required enough；
Wherein, the single microsatellite locus genotype extraction module obtains comparing logarithm for pre-processing sequencing data
According to, then comparison data is scanned, obtains the genotype of each microsatellite locus, the genotype tool of each microsatellite locus
Body includes the position that microsatellite locus place refers to genome, repetitive unit, the number of repetition on reference genome, sequencing number
The position repetitive unit number of repetition, corresponding read number and other optional informations are detected in；
Removal verification and measurement ratio is unsatisfactory for desired site module and is used to carry out microsatellite locus genotype to all training samples to mention
It takes, obtains the genotype of all training samples；Then filtering is unsatisfactory for the microsatellite locus of verification and measurement ratio requirement；
The higher site module of removal crowd's diversity is used for some microsatellite locus for some sample, is repeated with microsatellite
Cell-average number of repetition replaces the genotype of the microsatellite locus in the sample；Then it for each microsatellite locus, calculates
The mean μ and standard deviation sigma of sample microsatellite genotype；Multiplicity of the statistic being made of mean value and standard deviation to microsatellite locus
Property is assessed；Assessment result is carried out according to diversity, and screens microsatellite locus using diversity；
The small site module of discrimination is removed for calculating the microsatellite locus in the microsatellite instability positive and negative sample
Stability difference, then screening obtains effective detection site.
3. a kind of microsatellite instability detection method based on gene order-checking, which is characterized in that including microsatellite instability
Property detection method be broadly divided into microsatellite detection site selection and microsatellite instability detect two processes, it is specific as follows:
Step 1: microsatellite detection site selects: according to the sequencing data to certain tumor sample, effective detection site is selected,
Calculate the corresponding single instable threshold value of microsatellite locus of effective detection site and certain tumor sample microsatellite instability
Qualitative evaluation criterion；
Step 2: microsatellite instability detects: the corresponding single microsatellite position of effective detection site obtained according to step 1
The evaluation criterion of the instable threshold value of point and certain tumor sample microsatellite instability carries out microsatellite to detection sample
Unstability detection.
4. a kind of microsatellite instability detection method based on gene order-checking according to claim 3, feature exist
In selecting effective detection site, detailed process is as follows:
1) single microsatellite locus genotype is extracted: being pre-processed first to sequencing data, is obtained comparison data, then compare
Data are scanned, the genotype of each microsatellite locus is obtained, the genotype of each microsatellite locus specifically includes micro- defend
Refer to the position of genome where championship point, repetitive unit, the number of repetition on reference genome detects in sequencing data
The position repetitive unit number of repetition, corresponding read number and other optional informations；
2) removal verification and measurement ratio is unsatisfactory for desired site: carrying out the extraction of microsatellite locus genotype to all training samples, obtains
To the genotype of all training samples；Then filtering is unsatisfactory for the microsatellite locus of verification and measurement ratio requirement；
3) for some microsatellite locus of some sample, being averaged that number of repetition replaces with microsatellite repetitive unit should in the sample
The genotype of microsatellite locus；Then for each microsatellite locus, the mean μ and standard deviation of sample microsatellite genotype are calculated
σ；The statistic being made of mean value with standard deviation assesses the diversity of microsatellite locus；Assessment knot is carried out according to diversity
Fruit, and microsatellite locus is screened using diversity；
4) the microsatellite locus stability difference in the microsatellite instability positive and negative sample is calculated, then screening is had
Imitate detection site.
5. a kind of microsatellite instability detection method based on gene order-checking according to claim 4, feature exist
In, in step 2), filtering microsatellite locus of the verification and measurement ratio less than 50%；
In step 3), the microsatellite locus of σ/μ > 1 is removed when screening microsatellite locus using diversity.
6. a kind of microsatellite instability detection method based on gene order-checking according to claim 4, feature exist
In in step 4), then calculating microsatellite locus stability difference in the microsatellite instability positive and negative sample screens
Obtaining effective detection site, detailed process is as follows:
4.1) evaluation method of single microsatellite stability is determined, detailed process is as follows:
When defect occurs for intracellular mismatch repair system, mistake occurs in duplication and accumulates by intracellular DNA, if mould
Plate chain occurs to fold the variation that the same position DNA is then deleted in then subsequent progeny cell, if synthesizing chain
It folds, then insertion variation can occur for the same position DNA in the progeny cell of back；It, will in order to detect microsatellite instability
The process simulation of DNA cloning is a multinomial distribution；Detailed process is as follows:
Assuming that in microsatellite region, DNA during synthesis, each base or repetitive unit synthesis process be it is independent,
Fold will not occur simultaneously for the same position, template strand and synthesis chain；Template when each base or each unit replicate
Chain folding is event D, and the probability occurred is p；Synthesis chain folding is event I, probability q；The situation template chain and synthesis
Chain is not folded into event N, probability 1-p-q；Assuming that DNA microsatellite region base or repetition in a replication process
Unit number is L, and duplication produces m copy altogether, and the number that wherein event D occurs is d, and the number that event I occurs is i, thing
The probability that part N occurs is n, then d+i+n=mL；
Had based on above-mentioned hypothesis:
P in above formula, q pass through d, i, n estimation；
For each detected microsatellite locus, the stability of single microsatellite is measured according to p and/or q；
4.2) according to the single microsatellite locus method for estimating stability of step 4.1), microsatellite locus in all samples is calculated
Stability；For each microsatellite locus, examine the site stability whether in microsatellite feminine gender using non-parametric test method
There is significant difference in sample and in positive sample；Then all microsatellite locus are examined, are removed in negative sample and the positive
The microsatellite locus being not significantly different in sample obtains effective detection site.
7. a kind of microsatellite instability detection method based on gene order-checking according to claim 6, feature exist
In in step 1, calculating the corresponding single instable threshold value of microsatellite locus of effective detection site, detailed process is as follows:
For each effective detection site, using the evaluation method of order microsatellite stability calculates microsatellite really in step 4.1)
Stability in unstability negative sample, and the mean value and variance of computational stability, by stability mean value and three times variance
With the threshold value as the microsatellite locus stability.
8. a kind of microsatellite instability detection method based on gene order-checking according to claim 4, feature exist
In in step 1, determining the evaluation criterion of certain tumor sample microsatellite instability, detailed process is as follows: according to effective inspection
The threshold value of the corresponding microsatellite locus stability of location point, judges the stability of microsatellite each in sample, then root
Sample overall stability is assessed according to the stability of effective detection sites all in sample, determines sample entirety microsatellite not
Detection of Stability evaluation criterion.
9. a kind of microsatellite instability detection method based on gene order-checking according to claim 3, feature exist
In in step 1, calculating the stability of all effective detection sites in each sample, and calculate unstable anchor point and account for all detections
To the ratio in effective site, then sample microsatellite instability is judged according to ratio, is chosen at sensibility and special
Property value when reaching maximum be certain tumor sample microsatellite instability evaluation criterion, when test sample is greater than the evaluation mark
It is positive for microsatellite instability on time, it is otherwise microsatellite instability feminine gender.
10. a kind of microsatellite instability detection method based on gene order-checking according to claim 3, feature exist
In detailed process is as follows for step 2: first according to the set of the effective detection site of microsatellite instability, scanning sequencing number
According to the genotype for obtaining each microsatellite locus is distributed, and then calculates the stability of single microsatellite locus and according to single micro-
The stability of the single microsatellite of the corresponding threshold decision of the stability in satellite site, certain last tumor sample microsatellite instability
The evaluation criterion of property, judges sample microsatellite instability, obtains the microsatellite stability result of sample entirety.
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